Methods of upmodulating adaptive immune response using anti-pd1 antibodies

ABSTRACT

This disclosure provides antibodies and antigen-binding fragments that can act as agonists and/or antagonists of PD-1 (Programmed Death 1), thereby modulating immune responses in general, and those mediated by TcR and CD28, in particular. The disclosed compositions and methods may be used for example, in treating autoimmune diseases, inflammatory disorders, allergies, transplant rejection, cancer, and other immune system disorders.

RELATED APPLICATIONS

This application is a continuation of and claims priority under 35 USC§120 of U.S. patent application Ser. No. 10/540,084, filed Apr. 7, 2006,which is now allowed, which is a national stage application of andclaims priority under 35 USC §371 to PCT Application Serial No.PCT/IB2003/006304, filed Dec. 22. 2003, which claims the benefit of U.S.Provisional Application Ser. No. 60/45,354, filed Dec. 23, 2002, all ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The technical field relates to modulation of immune responses regulatedby the Programmed Death 1 (PD-1) receptor.

BACKGROUND OF THE INVENTION

An adaptive immune response involves activation, selection, and clonalproliferation of two major classes of lymphocytes termed T cells and Bcells. After encountering an antigen, T cells proliferate anddifferentiate into antigen-specific effector cells, while B cellsproliferate and differentiate into antibody-secreting cells.

T cell activation is a multi-step process requiring several signalingevents between the T cell and an antigen-presenting cell (APC). For Tcell activation to occur, two types of signals must be delivered to aresting T cell. The first type is mediated by the antigen-specific Tcell receptor (TcR), and confers specificity to the immune response. Thesecond, costimulatory, type regulates the magnitude of the response andis delivered through accessory receptors on the T cell.

A primary costimulatory signal is delivered through the activating CD28receptor upon engagement of its ligands B7-1 or B7-2. In contrast,engagement of the inhibitory CTLA-4 receptor by the same B7-1 or B7-2ligands results in attenuation of T cell response. Thus, CTLA-4 signalsantagonize costimulation mediated by CD28. At high antigenconcentrations, CD28 costimulation overrides the CTLA-4 inhibitoryeffect. Temporal regulation of the CD28 and CTLA-4 expression maintainsa balance between activating and inhibitory signals and ensures thedevelopment of an effective immune response, while safeguarding againstthe development of autoimmunity.

Molecular homologues of CD28 and CTLA-4 and their B-7 like ligands havebeen recently identified. ICOS is a CD28-like costimulatory receptor.PD-1 (Programmed Death 1) is an inhibitory receptor and a counterpart ofCTLA-4. This disclosure relates to modulation of immune responsesmediated by the PD-1 receptor.

PD-1 is a 50-55 kDa type I transmembrane receptor that was originallyidentified in a T cell line undergoing activation-induced apoptosis.PD-1 is expressed on T cells, B cells, and macrophages. The ligands forPD-1 are the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC).

PD-1 is a member of the immunoglobulin (Ig) superfamily that contains asingle Ig V-like domain in its extracellular region. The PD-1cytoplasmic domain contains two tyrosines, with the mostmembrane-proximal tyrosine (VAYEEL in mouse PD-1) located within an ITIM(immuno-receptor tyrosine-based inhibitory motif). The presence of anITIM on PD-1 indicates that this molecule functions to attenuate antigenreceptor signaling by recruitment of cytoplasmic phosphatases. Human andmurine PD-1 proteins share about 60% amino acid identity withconservation of four potential N-glycosylation sites, and residues thatdefine the Ig-V domain. The ITIM in the cytoplasmic region and theITIM-like motif surrounding the carboxy-terminal tyrosine (TEYATI inhuman and mouse) are also conserved between human and murineorthologues.

PD-1 is expressed on activated T cells, B cells, and monocytes.Experimental data implicates the interactions of PD-1 with its ligandsin downregulation of central and peripheral immune responses. Inparticular, proliferation in wild-type T cells but not in PD-1-deficientT cells is inhibited in the presence of PD-L1. Additionally,PD-1-deficient mice exhibit an autoimmune phenotype. PD-1 deficiency inthe C57BL/6 mice results in chronic progressive lupus-likeglomerulonephritis and arthritis. In Balb/c mice, PD-1 deficiency leadsto severe cardiomyopathy due to the presence of heart-tissue-specificself-reacting antibodies.

In general, a need exists to provide safe and effective therapeuticmethods for immune disorders such as, for example, autoimmune diseases,inflammatory disorders, allergies, transplant rejection, cancer, immunedeficiency, and other immune system-related disorders. Modulation of theimmune responses involved in these disorders can be accomplished bymanipulation of the PD-1 pathway.

SUMMARY OF THE INVENTION

The present disclosure provides antibodies that can act as agonistsand/or antagonists of PD-1, thereby modulating immune responsesregulated by PD-1. The disclosure further provides anti-PD-1 antibodiesthat comprise novel antigen-binding fragments. Anti-PD-1 antibodies ofthe invention are capable of (a) specifically binding to PD-1, includinghuman PD-1; (b) blocking PD-1 interactions with its natural ligand(s);or (c) performing both functions. Furthermore, the antibodies maypossess immunomodulatory properties, i.e., they may be effective inmodulating the PD-1-associated downregulation of immune responses.Depending on the method of use and the desired effect, the antibodiesmay be used to either enhance or inhibit immune responses.

Nonlimiting illustrative embodiments of the antibodies are referred toas PD1-17, PD1-28, PD1-33, PD1-35, and PD1-F2. Other embodimentscomprise a V_(H) and/or V_(L) domain of the Fv fragment of PD1-17,PD1-28, PD1-33, PD1-35, or PD1-F2. Further embodiments comprise one ormore complementarity determining regions (CDRs) of any of these V_(H)and V_(L) domains. Other embodiments comprise an H3 fragment of theV_(H) domain of PD1-17, PD1-28, PD1-33, PD1-35, or PD1-F2.

The disclosure also provides compositions comprising PD-1 antibodies,and their use in methods of modulating immune response, includingmethods of treating humans or animals. In particular embodiments,anti-PD-1 antibodies are used to treat or prevent immune disorders byvirtue of increasing or reducing the T cell response mediated byTcR/CD28. Disorders susceptible to treatment with compositions of theinvention include but are not limited to rheumatoid arthritis, multiplesclerosis, inflammatory bowel disease, Crohn's disease, systemic lupuserythematosis, type 1 diabetes, transplant rejection, graft-versus-hostdisease, hyperproliferative immune disorders, cancer, and infectiousdiseases.

Additionally, anti-PD-1 antibodies may be used diagnostically to detectPD-1 or its fragments in a biological sample. The amount of PD-1detected may be correlated with the expression level of PD-1, which, inturn, is correlated with the activation status of immune cells (e.g.,activated T cells, B cells, and monocytes) in the subject.

The disclosure also provides isolated nucleic acids, which comprise asequence encoding a V_(H) or V_(L) domain from the Fv fragment ofPD1-17, PD1-28, PD1-33, PD1-35, or PD1-F2. Also provided are isolatednucleic acids, which comprise a sequence encoding one or more CDRs fromany of the presently disclosed V_(H) and V_(L) domains. The disclosurealso provides vectors and host cells comprising such nucleic acids.

The disclosure further provides a method of producing new V_(H) andV_(L) domains and/or functional antibodies comprising all or a portionof such domains derived from the V_(H) or V_(L) domains of PD1-17,PD1-28, PD1-33, PD1-35, or PD1-F2.

Additional aspects of the disclosure will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practicing the invention. Theinvention is set forth and particularly pointed out in the appendedclaims, and the present disclosure should not be construed as limitingthe scope of the claims in any way. The following detailed descriptionincludes exemplary representations of various embodiments of theinvention, which are not restrictive of the invention, as claimed. Theaccompanying figures constitute a part of this specification and,together with the description, serve only to illustrate variousembodiments and not limit the invention. Citation of references is notan admission that these references are prior art to the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show reactivity of scFv antibodies with human PD-1 asdetermined by phage ELISA.

FIGS. 2A-2C show reactivity of IgG-converted antibodies with human ormouse PD-1 as determined by ELISA.

FIG. 3 shows results of an ELISA demonstrating that selected PD-1antibodies inhibit binding of PD-L1 to PD-1.

FIG. 4 shows results of an ELISA demonstrating that immunomodulatoryPD-1 antibodies bind to distinct sites on PD-1 as determined bycross-blocking ELISA assays.

FIG. 5 shows results of T-cell proliferation assays demonstrating thatco-engagement by TcR and anti-PD-1 antibody PD1-17 or PD-L1.Fc reducesproliferation. Co-engagement by TcR and anti-PD-1 J 110 has no effect onproliferation.

FIG. 6 demonstrates enhanced proliferation of primary T cells by PD1-17in a soluble form.

DETAILED DESCRIPTION Definitions

The term “antibody,” as used in this disclosure, refers to animmunoglobulin or a fragment or a derivative thereof, and encompassesany polypeptide comprising an antigen-binding site, regardless whetherit is produced in vitro or in vivo. The term includes, but is notlimited to, polyclonal, monoclonal, monospecific, polyspecific,non-specific, humanized, single-chain, chimeric, synthetic, recombinant,hybrid, mutated, and grafted antibodies. Unless otherwise modified bythe term “intact,” as in “intact antibodies,” for the purposes of thisdisclosure, the term “antibody” also includes antibody fragments such asFab, F(ab′)₂, Fv, scFv, Fd, dAb, and other antibody fragments thatretain antigen-binding function, i.e., the ability to bind PD-1specifically. Typically, such fragments would comprise anantigen-binding domain.

The terms “antigen-binding domain,” “antigen-binding fragment,” and“binding fragment” refer to a part of an antibody molecule thatcomprises amino acids responsible for the specific binding between theantibody and the antigen. In instances, where an antigen is large, theantigen-binding domain may only bind to a part of the antigen. A portionof the antigen molecule that is responsible for specific interactionswith the antigen-binding domain is referred to as “epitope” or“antigenic determinant.”

An antigen-binding domain typically comprises an antibody light chainvariable region (V_(L)) and an antibody heavy chain variable region(V_(H)), however, it does not necessarily have to comprise both. Forexample, a so-called Fd antibody fragment consists only of a V_(H)domain, but still retains some antigen-binding function of the intactantibody.

The term “repertoire” refers to a genetically diverse collection ofnucleotides derived wholly or partially from sequences that encodeexpressed immunoglobulins. The sequences are generated by in vivorearrangement of, e.g., V, D, and J segments for H chains and, e.g., Vand J segment for L chains. Alternatively, the sequences may begenerated from a cell line by in vitro stimulation, in response to whichthe rearrangement occurs. Alternatively, part or all of the sequencesmay be obtained by combining, e.g., unrearranged V segments with D and Jsegments, by nucleotide synthesis, randomised mutagenesis, and othermethods, e.g., as disclosed in U.S. Pat. No. 5,565,332.

The terms “specific interaction” and “specific binding” refer to twomolecules forming a complex that is relatively stable under physiologicconditions. Specific binding is characterized by a high affinity and alow to moderate capacity as distinguished from nonspecific binding whichusually has a low affinity with a moderate to high capacity. Typically,binding is considered specific when the affinity constant K_(A) ishigher than 10⁶ M⁻¹, or more preferably higher than 10⁸ M⁻¹. Ifnecessary, non-specific binding can be reduced without substantiallyaffecting specific binding by varying the binding conditions. Theappropriate binding conditions such as concentration of antibodies,ionic strength of the solution, temperature, time allowed for binding,concentration of a blocking agent (e.g., serum albumin, milk casein),etc., may be optimized by a skilled artisan using routine techniques.Illustrative conditions are set forth in Examples 1, 2, 4, 6 and 7.

The phrase “substantially as set out” means that the relevant CDR,V_(H), or V_(L) domain of the invention will be either identical to orhave only insubstantial differences in the specified regions (e.g., aCDR), the sequence of which is set out. Insubstantial differencesinclude minor amino acid changes, such as substitutions of 1 or 2 out ofany 5 amino acids in the sequence of a specified region.

The term “PD-1 activity” refers to one or more immunoregulatoryactivities associated with PD-1. For example, PD-1 is a negativeregulator of the TcR/CD28-mediated immune response. Procedures forassessing the PD-1 activity in vivo and in vitro are described inExamples 8, 9 and 10.

The terms “modulate,” “immunomodulatory,” and their cognates refer to areduction or an increase in the activity of PD-1 associated withdownregulation of T cell responses due to its interaction with ananti-PD-1 antibody, wherein the reduction or increase is relative to theactivity of PD-1 in the absence of the same antibody. A reduction or anincrease in activity is preferably at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or more. When PD-1 activity is reduced, theterms “modulatory” and “modulate” are interchangeable with the terms“inhibitory” and “inhibit.” When PD-1 activity is increased, the terms“modulatory” and “modulate” are interchangeable with the terms“activating” and “activate.” The activity of PD-1 can be determinedquantitatively using T cell proliferation assays as described inExamples 8 and 9.

The terms “treatment” and “therapeutic method” refer to both therapeutictreatment and prophylactic/preventative measures. Those in need oftreatment may include individuals already having a particular medicaldisorder as well as those who may ultimately acquire the disorder (i.e.,those needing preventative measures).

The term “effective amount” refers to a dosage or amount that issufficient to reduce the activity of PD-1 to result in amelioration ofsymptoms in a patient or to achieve a desired biological outcome, e.g.,increased cytolytic activity of T cells, induction of immune tolerance,reduction or increase of the PD-1 activity associated with the negativeregulation of T-cell mediated immune response, etc.

The term “isolated” refers to a molecule that is substantially free ofits natural environment. For instance, an isolated protein issubstantially free of cellular material or other proteins from the cellor tissue source from which it is derived. The term “isolated” alsorefers to preparations where the isolated protein is sufficiently pureto be administered as a pharmaceutical composition, or at least 70-80%(w/w) pure, more preferably, at least 80-90% (w/w) pure, even morepreferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%,98%, 99%, or 100% (w/w) pure.

Anti-PD-1 Antibodies

The disclosure provides anti-PD-1 antibodies that comprise novelantigen-binding fragments.

In general, antibodies can be made, for example, using traditionalhybridoma techniques (Kohler and Milstein (1975) Nature, 256: 495-499),recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage displayperformed with antibody libraries (Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597). For otherantibody production techniques, see also Antibodies: A LaboratoryManual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. Theinvention is not limited to any particular source, species of origin,method of production.

Intact antibodies, also known as immunoglobulins, are typicallytetrameric glycosylated proteins composed of two light (L) chains ofapproximately 25 kDa each and two heavy (H) chains of approximately 50kDa each. Two types of light chain, designated as the λ chain and the κchain, are found in antibodies. Depending on the amino acid sequence ofthe constant domain of heavy chains, immunoglobulins can be assigned tofive major classes: A, D, E, G and M, and several of these may befurther divided into subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂.

The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known in the art. For a review ofantibody structure, see Harlow et al., supra. Briefly, each light chainis composed of an N-terminal variable domain (V_(L)) and a constantdomain (C_(L)). Each heavy chain is composed of an N-terminal variabledomain (V_(H)), three or four constant domains (C_(H)), and a hingeregion. The C_(H) domain most proximal to V_(H) is designated as C_(H)1.The V_(H) and V_(L) domains consist of four regions of relativelyconserved sequence called framework regions (FR1, FR2, FR3, and FR4),which form a scaffold for three regions of hypervariable sequence calledcomplementarity determining regions (CDRs). The CDRs contain most of theresidues responsible for specific interactions with the antigen. Thethree CDRs are referred to as CDR1, CDR2, and CDR3. CDR constituents onthe heavy chain are referred to as H1, H2, and H3, while CDRconstituents on the light chain are referred to as L1, L2, and L3,accordingly. CDR3 and, particularly H3, are the greatest source ofmolecular diversity within the antigen-binding domain. H3, for example,can be as short as two amino acid residues or greater than 26.

The Fab fragment (Fragment antigen-binding) consists of the V_(H)-C_(H)1and V_(L)-C_(L) domains covalently linked by a disulfide bond betweenthe constant regions. To overcome the tendency of non-covalently linkedV_(H) and V_(L) domains in the Fv to dissociate when co-expressed in ahost cell, a so-called single chain (sc) Fv fragment (scFv) can beconstructed. In a scFv, a flexible and adequately long polypeptide linkseither the C-terminus of the V_(H) to the N-terminus of the V_(L) or theC-terminus of the V_(L) to the N-terminus of the V_(H). Most commonly, a15-residue (Gly₄Ser)₃ peptide is used as a linker but other linkers arealso known in the art.

Antibody diversity is a result of combinatorial assembly of multiplegermline genes encoding variable regions and a variety of somaticevents. The somatic events include recombination of variable genesegments with diversity (D) and joining (J) gene segments to make acomplete V_(H) region and the recombination of variable and joining genesegments to make a complete V_(L) region. The recombination processitself is imprecise, resulting in the loss or addition of amino acids atthe V(D)J junctions. These mechanisms of diversity occur in thedeveloping B cell prior to antigen exposure. After antigenicstimulation, the expressed antibody genes in B cells undergo somaticmutation.

Based on the estimated number of germline gene segments, the randomrecombination of these segments, and random V_(H)-V_(L) pairing, up to1.6×10⁷ different antibodies could be produced (Fundamental Immunology,3^(rd) ed., ed. Paul, Raven Press, New York, N.Y., 1993). When otherprocesses which contribute to antibody diversity (such as somaticmutation) are taken into account, it is thought that upwards of 1×10¹⁰different antibodies could be potentially generated (ImmunoglobulinGenes, 2^(nd) ed., eds. Jonio et al., Academic Press, San Diego, Calif.,1995). Because of the many processes involved in antibody diversity, itis highly unlikely that independently generated antibodies will haveidentical or even substantially similar amino acid sequences in theCDRs.

The disclosure provides novel CDRs derived from human immunoglobulingene libraries. The structure for carrying a CDR will generally be anantibody heavy or light chain or a portion thereof, in which the CDR islocated at a location corresponding to the CDR of naturally occurringV_(H) and V_(L). The structures and locations of immunoglobulin variabledomains may be determined, for example, as described in Kabat et al.,Sequences of Proteins of Immunological Interest, No. 91-3242, NationalInstitutes of Health Publications, Bethesda, Md., 1991.

DNA and amino acid sequences of anti-PD-1 antibodies, their scFvfragment, V_(H) and V_(L) domains, and CDRs are set forth in theSequence Listing and are enumerated as listed in Table 1. Particularnonlimiting illustrative embodiments of the antibodies are referred toas PD1-17, PD1-28, PD1-33, PD1-35, and PD1-F2. The positions for eachCDR within the V_(H) and V_(L) domains of the illustrative embodimentsare listed in Tables 2 and 3.

TABLE 1 DNA and Amino Acid (AA) Sequences of V_(H) and V_(L) Domains andCDRs Sequence PD1-17 PD1-28 PD1-33 PD1-35 PD1-F2 V_(H) DNA SEQ ID NO: 1SEQ ID NO: 5 SEQ ID NO: 9 SEQ ID NO: 13 SEQ ID NO: 46 V_(H) AA SEQ IDNO: 2 SEQ ID NO: 6 SEQ ID NO: 10 SEQ ID NO: 14 SEQ ID NO: 47 V_(L) DNASEQ ID NO: 3 SEQ ID NO: 7 SEQ ID NO: 11 SEQ ID NO: 15 SEQ ID NO: 48V_(L) AA SEQ ID NO: 4 SEQ ID NO: 8 SEQ ID NO: 12 SEQ ID NO: 16 SEQ IDNO: 49 H1 AA SEQ ID NO: 17 SEQ ID NO: 23 SEQ ID NO: 29 SEQ ID NO: 35 SEQID NO: 50 H2 AA SEQ ID NO: 18 SEQ ID NO: 24 SEQ ID NO: 30 SEQ ID NO: 36SEQ ID NO: 51 H3 AA SEQ ID NO: 19 SEQ ID NO: 25 SEQ ID NO: 31 SEQ ID NO:37 SEQ ID NO: 52 L1 AA SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 32 SEQ IDNO: 38 SEQ ID NO: 53 L2 AA SEQ ID NO: 21 SEQ ID NO: 27 SEQ ID NO: 33 SEQID NO: 39 SEQ ID NO: 54 L3 AA SEQ ID NO: 22 SEQ ID NO: 28 SEQ ID NO: 34SEQ ID NO: 40 SEQ ID NO: 55

TABLE 2 Positions of Heavy Chain CDRs PD1-17 PD1-28 PD1-33 PD1-35 PD1-F2CDR SEQ ID NO: 2 SEQ ID NO: 6 SEQ ID NO: 10 SEQ ID NO: 14 SEQ ID NO: 47H1 31-42 31-35 31-35 31-37 34-42 H2 57-72 50-66 50-66 52-67 57-73 H3105-117  99-108  99-108 100-116 106-114

TABLE 3 Positions of Light Chain CDRs PD1-17 PD1-28 PD1-33 PD1-35 PD1-F2CDR SEQ ID NO: 4 SEQ ID NO: 8 SEQ ID NO: 12 SEQ ID NO: 16 SEQ ID NO: 49L1 23-35 23-33 23-36 23-35 28-35 L2 51-57 49-55 52-58 51-57 54-61 L3 92-100 88-98  91-102  90-100  94-101

Anti-PD-1 antibodies may optionally comprise antibody constant regionsor parts thereof. For example, a V_(L) domain may have attached, at itsC terminus, antibody light chain constant domains including human Cκ orCλ chains. Similarly, a specific antigen-binding domain based on a V_(H)domain may have attached all or part of an immunoglobulin heavy chainderived from any antibody isotope, e.g., IgG, IgA, IgE and IgM and anyof the isotope sub-classes, which include but are not limited to, IgG₁and IgG₄. In the exemplary embodiments, PD1-17, PD1-28, PD1-33, andPD1-35, antibodies comprise C-terminal fragments of heavy and lightchains of human IgG_(1λ), while PD1-F2 comprises C-terminal fragments ofheavy and light chains of human IgG_(1κ). The DNA and amino acidsequences for the C-terminal fragment of are well known in the art (see,e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No.91-3242, National Institutes of Health Publications, Bethesda, Md.,1991). Nonlimiting exemplary sequences are set forth in Table 4.

TABLE 4 C-Terminal Region DNA Amino acid IgG1 heavy chain SEQ ID NO: 44SEQ ID NO: 45 λ light chain SEQ ID NO: 42 SEQ ID NO: 43 κ light chainSEQ ID NO: 57 SEQ ID NO: 58

Certain embodiments comprise a V_(H) and/or V_(L) domain of an Fvfragment from PD1-17, PD1-28, PD1-33, PD1-35, and PD1-F2. Furtherembodiments comprise at least one CDR of any of these V_(H) and V_(L)domains. Antibodies, comprising at least one of the CDR sequences setout in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NOs:16-40, SEQ ID NO:47, or SEQ IDNO:49 are encompassed within the scope of this invention. An embodiment,for example, comprises an H3 fragment of the V_(H) domain of antibodieschosen from at least one of PD1-17, PD1-28, PD1-33, PD1-35, and PD1-F2.

In certain embodiments, the V_(H) and/or V_(L) domains may be germlined,i.e., the framework regions (FRs) of these domains are mutated usingconventional molecular biology techniques to match those produced by thegermline cells. In other embodiments, the framework sequences remaindiverged from the consensus germline sequences.

In certain embodiments, the antibodies specifically bind an epitopewithin the extracellular domain of human PD-1. The predictedextracellular domain consists of a sequence from about amino acid 21 toabout amino acid 170 of SEQ ID NO:41 (Swissport Accession No. Q15116).In certain other embodiments, the antibodies specifically bind anepitope within the extracellular domain of mouse PD-1, with an affinityof more than 10⁷ M⁻¹, and preferably more than 10⁸ M⁻¹. The amino acidsequence of mouse PD-1 is set out in SEQ ID NO:56 (Accession No.NM_(—)008798) and is as a whole about 60% identical to its humancounterpart. In further embodiments, antibodies of the invention bind tothe PD-L-binding domain of PD-1.

It is contemplated that antibodies of the invention may also bind withother proteins, including, for example, recombinant proteins comprisingall or a portion of the PD-1 extracellular domain.

One of ordinary skill in the art will recognize that the antibodies ofthis invention may be used to detect, measure, and inhibit proteins thatdiffer somewhat from PD-1. The antibodies are expected to retain thespecificity of binding so long as the target protein comprises asequence which is at least about 60%, 70%, 80%, 90%, 95% or moreidentical to any sequence of at least 100, 80, 60, 40 or 20 ofcontiguous amino acids in the sequence set forth SEQ ID NO:41. Thepercent identity is determined by standard alignment algorithms such as,for example, Basic Local Alignment Tool (BLAST) described in Altshul etal. (1990) J. Mol. Biol., 215: 403-410, the algorithm of Needleman etal. (1970) J. Mol. Biol., 48: 444-453, or the algorithm of Meyers et al.(1988) Comput. Appl. Biosci., 4: 11-17.

In addition to the sequence homology analyses, epitope mapping (see,e.g., Epitope Mapping Protocols, ed. Morris, Humana Press, 1996) andsecondary and tertiary structure analyses can be carried out to identifyspecific 3D structures assumed by the disclosed antibodies and theircomplexes with antigens. Such methods include, but are not limited to,X-ray crystallography (Engstom (1974) Biochem. Exp. Biol., 11: 7-13) andcomputer modeling of virtual representations of the presently disclosedantibodies (Fletterick et al. (1986) Computer Graphics and MolecularModeling, in Current Communications in Molecular Biology, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.).

Derivatives

This disclosure also provides a method for obtaining an antibodyspecific for PD-1. CDRs in such antibodies are not limited to thespecific sequences of V_(H) and V_(L) identified in Table 1 and mayinclude variants of these sequences that retain the ability tospecifically bind PD-1. Such variants may be derived from the sequenceslisted in Table 1 by a skilled artisan using techniques well known inthe art. For example, amino acid substitutions, deletions, or additions,can be made in the FRs and/or in the CDRs. While changes in the FRs areusually designed to improve stability and immunogenicity of theantibody, changes in the CDRs are typically designed to increaseaffinity of the antibody for its target. Variants of FRs also includenaturally occurring immunoglobulin allotypes. Such affinity-increasingchanges may be determined empirically by routine techniques that involvealtering the CDR and testing the affinity antibody for its target. Forexample, conservative amino acid substitutions can made within any oneof the disclosed CDRs. Various alterations can be made according to themethods described in Antibody Engineering, 2 ed., Oxford UniversityPress, ed. Borrebaeck, 1995. These include but are not limited tonucleotide sequences that are altered by the substitution of differentcodons that encode a functionally equivalent amino acid residue withinthe sequence, thus producing a “silent” change. For example, thenonpolar amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine and glutamin. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Substitutes for anamino acid within the sequence may be selected from other members of theclass to which the amino acid belongs (see Table 5). Furthermore, anynative residue in the polypeptide may also be substituted with alanine(see, e.g., MacLennan et al. (1998) Acta Physio. Scand. Suppl.643:55-67; Sasaki et al. (1998) Adv. Biophys. 35:1-24).

Derivatives and analogs of antibodies of the invention can be producedby various techniques well known in the art, including recombinant andsynthetic methods (Maniatis (1990) Molecular Cloning, A LaboratoryManual, 2 ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,and Bodansky et al. (1995) The Practice of Peptide Synthesis, 2^(nd)ed., Spring Verlag, Berlin, Germany).

TABLE 5 Original Exemplary Typical Residues Substitutions SubstitutionsAla (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln Gln Asp(D) Glu Glu Cys (C) Ser, Ala Ser Gln (Q) Asn Asn Gly (G) Pro, Ala AlaHis (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe,Norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K)Arg, 1,4-Diamino-butyric Acid, Gln, Asn Arg Met (M) Leu, Phe, Ile LeuPhe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Gly Ser (S) Thr, Ala,Cys Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, SerPhe Val (V) Ile, Met, Leu, Phe, Ala, Norleucine Leu

In one embodiment, a method for making a V_(H) domain which is an aminoacid sequence variant of a V_(H) domain of the invention comprises astep of adding, deleting, substituting, or inserting one or more aminoacids in the amino acid sequence of the presently disclosed V_(H)domain, optionally combining the V_(H) domain thus provided with one ormore V_(L) domains, and testing the V_(H) domain or V_(H)/V_(L)combination or combinations for a specific binding to PD-1 or and,optionally, testing the ability of such antigen-binding domain tomodulate PD-1 activity. The V_(L) domain may have an amino acid sequencethat is identical or is substantially as set out according to Table 1.

An analogous method can be employed in which one or more sequencevariants of a V_(L) domain disclosed herein are combined with one ormore V_(H) domains.

A further aspect of the disclosure provides a method of preparingantigen-binding fragment that specifically binds with PD-1. The methodcomprises:

(a) providing a starting repertoire of nucleic acids encoding a V_(H)domain that either includes a CDR3 to be replaced or lacks a CDR3encoding region;

(b) combining the repertoire with a donor nucleic acid encoding an aminoacid sequence substantially as set out herein for a V_(H) CDR3 (i.e.,H3) such that the donor nucleic acid is inserted into the CDR3 region inthe repertoire, so as to provide a product repertoire of nucleic acidsencoding a V_(H) domain;

(c) expressing the nucleic acids of the product repertoire;

(d) selecting a binding fragment specific for PD-1; and

(e) recovering the specific binding fragment or nucleic acid encodingit.

Again, an analogous method may be employed in which a V_(L) CDR3 (i.e.,L3) of the invention is combined with a repertoire of nucleic acidsencoding a V_(L) domain, which either include a CDR3 to be replaced orlack a CDR3 encoding region. The donor nucleic acid may be selected fromnucleic acids encoding an amino acid sequence substantially as set outin SEQ ID NO:17-40 or SEQ ID NO:50-55.

A sequence encoding a CDR of the invention (e.g., CDR3) may beintroduced into a repertoire of variable domains lacking the respectiveCDR (e.g., CDR3), using recombinant DNA technology, for example, usingmethodology described by Marks et al. (Bio/Technology (1992) 10:779-783). In particular, consensus primers directed at or adjacent tothe 5′ end of the variable domain area can be used in conjunction withconsensus primers to the third framework region of human V_(H) genes toprovide a repertoire of V_(H) variable domains lacking a CDR3. Therepertoire may be combined with a CDR3 of a particular antibody. Usinganalogous techniques, the CDR3-derived sequences may be shuffled withrepertoires of V_(H) or V_(L) domains lacking a CDR3, and the shuffledcomplete V_(H) or V_(L) domains combined with a cognate V_(L) or V_(H)domain to make the PD-1-specific antibodies of the invention. Therepertoire may then be displayed in a suitable host system such as thephage display system such as described in WO92/01047 so that suitableantigen-binding fragments can be selected.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer (Nature (1994) 370: 389-391), who describes the technique inrelation to a β-lactamase gene but observes that the approach may beused for the generation of antibodies.

In further embodiments, one may generate novel V_(H) or V_(L) regionscarrying one or more sequences derived from the sequences disclosedherein using random mutagenesis of one or more selected V_(H) and/orV_(L) genes. One such technique, error-prone PCR, is described by Gramet al. (Proc. Nat. Acad. Sci. U.S.A. (1992) 89: 3576-3580).

Another method that may be used is to direct mutagenesis to CDRs ofV_(H) or V_(L) genes. Such techniques are disclosed by Barbas et al.(Proc. Nat. Acad. Sci. U.S.A. (1994) 91: 3809-3813) and Schier et al.(J. Mol. Biol. (1996) 263: 551-567).

Similarly, one or more, or all three CDRs may be grafted into arepertoire of V_(H) or V_(L) domains, which are then screened for anantigen-binding fragment specific for PD-1.

A portion of an immunoglobulin variable domain will comprise at leastone of the CDRs substantially as set out herein and, optionally,intervening framework regions from the scFv fragments as set out herein.The portion may include at least about 50% of either or both of FR1 andFR4, the 50% being the C-terminal 50% of FR1 and the N-terminal 50% ofFR4. Additional residues at the N-terminal or C-terminal end of thesubstantial part of the variable domain may be those not normallyassociated with naturally occurring variable domain regions. Forexample, construction of antibodies by recombinant DNA techniques mayresult in the introduction of N- or C-terminal residues encoded bylinkers introduced to facilitate cloning or other manipulation steps.Other manipulation steps include the introduction of linkers to joinvariable domains to further protein sequences including immunoglobulinheavy chain constant regions, other variable domains (for example, inthe production of diabodies), or proteinaceous labels as discussed infurther detail below.

Although the embodiments illustrated in the Examples comprise a“matching” pair of V_(H) and V_(L) domains, a skilled artisan willrecognize that alternative embodiments may comprise antigen-bindingfragments containing only a single CDR from either V_(L) or V_(H)domain. Either one of the single chain specific binding domains can beused to screen for complementary domains capable of forming a two-domainspecific antigen-binding fragment capable of, for example, binding toPD-1. The screening may be accomplished by phage display screeningmethods using the so-called hierarchical dual combinatorial approachdisclosed in WO92/01047, in which an individual colony containing eitheran H or L chain clone is used to infect a complete library of clonesencoding the other chain (L or H) and the resulting two-chain specificbinding domain is selected in accordance with phage display techniquesas described.

Anti-PD1 antibodies described herein can be linked to another functionalmolecule, e.g., another peptide or protein (albumin, another antibody,etc.), toxin, radioisotope, cytotoxic or cytostatic agents. For example,the antibodies can be linked by chemical cross-linking or by recombinantmethods. The antibodies may also be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. No.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Theantibodies can be chemically modified by covalent conjugation to apolymer, for example, to increase their circulating half-life. Exemplarypolymers and methods to attach them are also shown in U.S. Pat. Nos.4,766,106; 4,179,337; 4,495,285 and 4,609,546.

The disclosed antibodies may also be altered to have a glycosylationpattern that differs from the native pattern. For example, one or morecarbohydrate moieties can be deleted and/or one or more glycosylationsites added to the original antibody. Addition of glycosylation sites tothe presently disclosed antibodies may be accomplished by altering theamino acid sequence to contain glycosylation site consensus sequencesknown in the art. Another means of increasing the number of carbohydratemoieties on the antibodies is by chemical or enzymatic coupling ofglycosides to the amino acid residues of the antibody. Such methods aredescribed in WO 87/05330 and in Aplin et al. (1981) CRC Crit. Rev.Biochem., 22: 259-306. Removal of any carbohydrate moieties from theantibodies may be accomplished chemically or enzymatically, for example,as described by Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259:52; and Edge et al. (1981) Anal. Biochem., 118: 131 and by Thotakura etal. (1987) Meth. Enzymol., 138: 350. The antibodies may also be taggedwith a detectable, or functional, label.

Detectable labels include radiolabels such as ¹³¹I or ⁹⁹Tc, which mayalso be attached to antibodies using conventional chemistry. Detectablelabels also include enzyme labels such as horseradish peroxidase oralkaline phosphatase. Detectable labels further include chemicalmoieties such as biotin, which may be detected via binding to a specificcognate detectable moiety, e.g., labeled avidin.

Antibodies, in which CDR sequences differ only insubstantially fromthose set out in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NOs:16-40, SEQ ID NO:47, orSEQ ID NO:49 are encompassed within the scope of this invention.Typically, an amino acid is substituted by a related amino acid havingsimilar charge, hydrophobic, or stereochemical characteristics. Suchsubstitutions would be within the ordinary skills of an artisan. Unlikein CDRs, more substantial changes can be made in FRs without adverselyaffecting the binding properties of an antibody. Changes to FRs include,but are not limited to, humanizing a non-human derived or engineeringcertain framework residues that are important for antigen contact or forstabilizing the binding site, e.g., changing the class or subclass ofthe constant region, changing specific amino acid residues which mightalter the effector function such as Fc receptor binding, e.g., asdescribed in U.S. Pat. Nos. 5,624,821 and 5,648,260 and Lund et al.(1991) J. Immun. 147: 2657-2662 and Morgan et al. (1995) Immunology 86:319-324, or changing the species from which the constant region isderived.

One of skill in the art will appreciate that the modifications describedabove are not all-exhaustive, and that many other modifications would beobvious to a skilled artisan in light of the teachings of the presentdisclosure.

Nucleic Acids, Cloning and Expression Systems

The present disclosure further provides isolated nucleic acids encodingthe disclosed antibodies. The nucleic acids may comprise DNA or RNA andmay be wholly or partially synthetic or recombinant. Reference to anucleotide sequence as set out herein encompasses a DNA molecule withthe specified sequence, and encompasses a RNA molecule with thespecified sequence in which U is substituted for T, unless contextrequires otherwise.

The nucleic acids provided herein comprise a coding sequence for a CDR,a V_(H) domain, and/or a V_(L) domain disclosed herein.

The present disclosure also provides constructs in the form of plasmids,vectors, phagemids, transcription or expression cassettes which compriseat least one nucleic acid encoding a CDR, a V_(H) domain, and/or a V_(L)domain disclosed here.

The disclosure further provides a host cell which comprises one or moreconstructs as above.

Also provided are nucleic acids encoding any CDR (H1, H2, H3, L1, L2, orL3), V_(H) or V_(L) domain, as well as methods of making of the encodedproducts. The method comprises expressing the encoded product from theencoding nucleic acid. Expression may be achieved by culturing underappropriate conditions recombinant host cells containing the nucleicacid. Following production by expression a V_(H) or V_(L) domain, orspecific binding member may be isolated and/or purified using anysuitable technique, then used as appropriate.

Antigen-binding fragments, V_(H) and/or V_(L) domains, and encodingnucleic acid molecules and vectors may be isolated and/or purified fromtheir natural environment, in substantially pure or homogeneous form,or, in the case of nucleic acid, free or substantially free of nucleicacid or genes of origin other than the sequence encoding a polypeptidewith the required function.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known in the art. For cells suitable forproducing antibodies, see Gene Expression Systems, Academic Press, eds.Fernandez et al., 1999. Briefly, suitable host cells include bacteria,plant cells, mammalian cells, and yeast and baculovirus systems.Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells, HeLacells, baby hamster kidney cells, NS0 mouse myeloma cells, and manyothers. A common bacterial host is E. coli. Any protein expressionsystem compatible with the invention may be used to produce thedisclosed antibodies. Suitable expression systems include transgenicanimals described in Gene Expression Systems, Academic Press, eds.Fernandez et al., 1999.

Suitable vectors can be chosen or constructed, so that they containappropriate regulatory sequences, including promoter sequences,terminator sequences, polyadenylation sequences, enhancer sequences,marker genes and other sequences as appropriate. Vectors may be plasmidsor viral, e.g., phage or phagemid, as appropriate. For further detailssee, for example, Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor Laboratory Press, 1989. Manyknown techniques and protocols for manipulation of nucleic acid, forexample, in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Current Protocols inMolecular Biology, 2 Edition, eds. Ausubel et al., John Wiley &; Sons,1992.

A further aspect of the disclosure provides a host cell comprising anucleic acid as disclosed here. A still further aspect provides a methodcomprising introducing such nucleic acid into a host cell. Theintroduction may employ any available technique. For eukaryotic cells,suitable techniques may include calcium phosphate transfection,DEAE-Dextran, electroporation, liposome-mediated transfection andtransduction using retrovirus or other virus, e.g., vaccinia or, forinsect cells, baculovirus. For bacterial cells, suitable techniques mayinclude calcium chloride transformation, electroporation andtransfection using bacteriophage. The introduction of the nucleic acidinto the cells may be followed by causing or allowing expression fromthe nucleic acid, e.g., by culturing host cells under conditions forexpression of the gene.

Methods of Use

The disclosed anti-PD-1 antibodies are capable of modulating thePD-1-associated downregulation of the immune responses. In particularembodiments, the immune response is TcR/CD28-mediated. The disclosedantibodies can act as either agonists or antagonists of PD-1, dependingon the method of their use. The antibodies can be used to prevent,diagnose, or treat medical disorders in mammals, especially, in humans.Antibodies of the invention can also be used for isolating PD-1 orPD-1-expressing cells. Furthermore, the antibodies can be used to treata subject at risk of or susceptible to a disorder or having a disorderassociated with aberrant PD-1 expression or function.

Antibodies of the invention can be used in methods for induction oftolerance to a specific antigen (e.g., a therapeutic protein). In oneembodiment, tolerance is induced against a specific antigen byco-administration of antigen and an anti-PD-1 antibody of the invention.For example, patients that received Factor VIII frequently generateantibodies to this protein; co-administration of an anti-PD-1 antibodyof the invention in combination with recombinant Factor VIII is expectedto result in the downregulation of immune responses to this clottingfactor.

Antibodies of the invention can be used in circumstances where areduction in the level of immune response may be desirable, for example,in certain types of allergy or allergic reactions (e.g., by inhibitionof IgE production), autoimmune diseases (e.g., rheumatoid arthritis,type I diabetes mellitus, multiple sclerosis, inflammatory boweldisease, Crohn's disease, and systemic lupus erythematosis), tissue,skin and organ transplant rejection and graft-versus-host disease(GVHD).

When diminished immune response is desirable, the anti-PD-1 antibodiesof the invention may be used as agonists to PD-1 in order to enhance thePD-1-associated attenuation of the immune response. In theseembodiments, co-presentation and physical proximity between positive(i.e., mediated by an antigen receptor, e.g., TcR or BcR) and negative(i.e., PD-1) signals are required. The preferred distance is less thanor comparable to the size of a naturally occurring antigen-presentingcell, i.e., less than about 100 μm; more preferably, less than about 50μm; and most preferably, less than about 20 μm.

In some embodiments, the positive (activating) and the negative(inhibiting) signals are provided by a ligand or antibodies immobilizedon solid support matrix, or a carrier. In various embodiments, the solidsupport matrix may be composed of polymer such as activated agarose,dextran, cellulose, polyvinylidene fluoride (PVDF). Alternatively, thesolid support matrix may be based on silica or plastic polymers, e.g.,as nylon, dacron, polystyrene, polyacrylates, polyvinyls, teflons, etc.

The matrix can be implanted into the spleen of a patient. Alternatively,the matrix may be used for the ex vivo incubation of T cells obtainedfrom a patient, which are then separated and implanted back into thepatient. The matrix may also be made from a biodegradable material suchpolyglycolic acid, polyhydroxyalkanoate, collagen or gelatin so thatthey can be injected into the patient's peritoneal cavity, and dissolveafter some time following the injection. The carrier can be shaped tomimic a cell (e.g., bead or micro sphere).

In some embodiments, the positive signal is delivered by aT-cell-activating anti-CD3 antibody, which binds TcR. Activatinganti-CD3 antibodies are known in the art (see, for example, U.S. Pat.Nos. 6,405,696 and 5,316,763). The ratio between the activating TcRsignal and negative PD-1 signal is determined experimentally usingconventional procedures known in the art or as described in Examples 8,9 and 10.

Under certain circumstances, it may be desirable to elicit or enhance apatient's immune response in order to treat an immune disorder orcancer. The disorders being treated or prevented by the disclosedmethods include but are not limited to infections with microbes (e.g.,bacteria), viruses (e.g., systemic viral infections such as influenza,viral skin diseases such as herpes or shingles), or parasites; andcancer (e.g., melanoma and prostate cancers).

Stimulation of T cell activation with anti-PD-1 antibodies enhances T-Tcell responses. In such cases, antibodies act as antagonists of PD-1.Thus, in some embodiments, the antibodies can be used to inhibit orreduce the downregulatory activity associated with PD-1, i.e., theactivity associated with downregulation of TcR/CD28-mediated immuneresponse. In these embodiments, the antibodies are not coupled to apositive signal such as the TcR-mediated stimulation, e.g., theantibodies are in their soluble, support-unbound, form. As demonstratedin the Examples, a blockade of PD-1/PD-L interaction with antagonizinganti-PD-1 antibodies leads to enhanced T cell proliferative responses,consistent with a downregulatory role for the PD-1 pathway in T-Tinteractions. In various embodiments, the antibodies inhibit binding ofPD-L to PD-1 with an IC₅₀ of less than 10 nM, and more preferably lessthen 5 nM, and most preferably less than 1 nM. Inhibition of PD-Lbinding can be measured as described in Example 6 or using techniquesknown in the art.

The antibodies or antibody compositions of the present invention areadministered in therapeutically effective amounts. Generally, atherapeutically effective amount may vary with the subject's age,condition, and sex, as well as the severity of the medical condition ofthe subject. A therapeutically effective amount of antibody ranges fromabout 0.001 to about 30 mg/kg body weight, preferably from about 0.01 toabout 25 mg/kg body weight, from about 0.1 to about 20 mg/kg bodyweight, or from about 1 to about 10 mg/kg. The dosage may be adjusted,as necessary, to suit observed effects of the treatment. The appropriatedose is chosen based on clinical indications by a treating physician.

The antibodies may be given as a bolus dose, to maximize the circulatinglevels of antibodies for the greatest length of time after the dose.Continuous infusion may also be used after the bolus dose.

Immune cells (e.g., activated T cells, B cells, or monocytes) can alsobe isolated from a patient and incubated ex vivo with antibodies of theinvention. In some embodiments, immune responses can be inhibited byremoving immune cells from a subject, contacting the immune cells invitro with an anti-PD-1 antibody of the invention concomitantly withactivation of the immune cells (e.g., by antibodies to the TcR and/orBcR antigen receptor). In such embodiments, the anti-PD-1 antibodyshould be used in a multivalent form such that PD-1 molecules on thesurface of an immune cell become “crosslinked” upon binding to suchantibodies. For example, the anti-PD-1 antibodies can be bound to solidsupport, such as beads, or crosslinked via a secondary antibody. Theimmune cells may be then isolated using methods known in the art andreimplanted into the patient.

In another aspect, the antibodies of the invention can be used as atargeting agent for delivery of another therapeutic or a cytotoxic agent(e.g., a toxin) to a cell expressing PD-1. The method includesadministering an anti-PD-1 antibody coupled to a therapeutic or acytotoxic agent or under conditions that allow binding of the antibodyto PD-1.

The antibodies of the invention may also be used to detect the presenceof PD-1 in biological samples. The amount of PD-1 detected may becorrelated with the expression level of PD-1, which, in turn, iscorrelated with the activation status of immune cells (e.g., activated Tcells, B cells, and monocytes) in the subject.

Detection methods that employ antibodies are well known in the art andinclude, for example, ELISA, radioimmunoassay, immunoblot, Western blot,immunofluorescence, immunoprecipitation. The antibodies may be providedin a diagnostic kit that incorporates one or more of these techniques todetect PD-1. Such a kit may contain other components, packaging,instructions, or other material to aid the detection of the protein.

Where the antibodies are intended for diagnostic purposes, it may bedesirable to modify them, for example, with a ligand group (such asbiotin) or a detectable marker group (such as a fluorescent group, aradioisotope or an enzyme). If desired, the antibodies of the inventionmay be labeled using conventional techniques. Suitable detectable labelsinclude, for example, fluorophores, chromophores, radioactive atoms,electron-dense reagents, enzymes, and ligands having specific bindingpartners. Enzymes are typically detected by their activity. For example,horseradish peroxidase can be detected by its ability to converttetramethylbenzidine (TMB) to a blue pigment, quantifiable with aspectrophotometer. For detection, suitable binding partners include, butare not limited to, biotin and avidin or streptavidin, IgG and proteinA, and the numerous receptor-ligand couples known in the art. Otherpermutations and possibilities will be readily apparent to those ofordinary skill in the art, and are considered as equivalents within thescope of the instant invention.

Antibodies of the invention can be used in screening methods to identifyinhibitors of the PD-1 pathway effective as therapeutics. In such ascreening assay, a first binding mixture is formed by combining PD-1 andan antibody of the invention; and the amount of binding in the firstbinding mixture (M₀) is measured. A second binding mixture is alsoformed by combining PD-1, the antibody, and the compound or agent to bescreened, and the amount of binding in the second binding mixture (M₁)is measured. A compound to be tested may be another anti-PD-1 antibody,as illustrated in the Examples. The amounts of binding in the first andsecond binding mixtures are then compared, for example, by calculatingthe M₁/M₀ ratio. The compound or agent is considered to be capable ofmodulating a PD-1-associated downregulation of immune responses if adecrease in binding in the second binding mixture as compared to thefirst binding mixture is observed. The formulation and optimization ofbinding mixtures is within the level of skill in the art, such bindingmixtures may also contain buffers and salts necessary to enhance or tooptimize binding, and additional control assays may be included in thescreening assay of the invention. Compounds found to reduce thePD-1-antibody binding by at least about 10% (i.e., M₁/M₀<0.9),preferably greater than about 30% may thus be identified and then, ifdesired, secondarily screened for the capacity to ameliorate a disorderin other assays or animal models as described below. The strength of thebinding between PD-1 and an antibody can be measured using, for example,an enzyme-linked immunoadsorption assay (ELISA), radio-immunoassay(RIA), surface plasmon resonance-based technology (e.g., Biacore), allof which are techniques well known in the art.

The compound may then be tested in vitro as described in the Examples orin an animal model (see, generally, Immunologic Defects in LaboratoryAnimals, eds. Gershwin et al., Plenum Press, 1981), for example, such asthe following: the SWR X NZB (SNF1) transgenic mouse model (Uner et al.(1998) J. Autoimmune. 11(3): 233-240), the KRN transgenic mouse (K/BxN)model (Ji et al. (1999) Immunol. Rev. 169: 139); NZB X NZW (B/W) mice, amodel for SLE (Riemekasten et al. (2001) Arthritis Rheum., 44(10):2435-2445); experimental autoimmune encephalitis (EAE) in mouse, a modelfor multiple sclerosis (Tuohy et al. (1988) J. Immunol. 141: 1126-1130,Sobel et al. (1984) J. Immunol. 132: 2393-2401, and Traugott, CellImmunol. (1989) 119: 114-129); the NOD mouse model of diabetes (Baxteret al. (1991) Autoimmunity, 9(1): 61-67), etc.).

Preliminary doses as, for example, determined according to animal tests,and the scaling of dosages for human administration is performedaccording to art-accepted practices. Toxicity and therapeutic efficacycan be determined by standard pharmaceutical procedures in cell culturesor experimental animals. The data obtained from the cell culture assaysor animal studies can be used in formulating a range of dosage for usein humans. Therapeutically effective dosages achieved in one animalmodel can be converted for use in another animal, including humans,using conversion factors known in the art (see, e.g., Freireich et al.(1966) Cancer Chemother. Reports, 50(4): 219-244).

Pharmaceutical Compositions and Methods of Administration

The disclosure provides compositions comprising anti-PD-1 antibodies.Such compositions may be suitable for pharmaceutical use andadministration to patients. The compositions typically comprise one ormore antibodies of the present invention and a pharmaceuticallyacceptable excipient. The phrase “pharmaceutically acceptable excipient”includes any and all solvents, dispersion media, coatings, antibacterialagents and antifungal agents, isotonic agents, and absorption delayingagents, and the like, that are compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. The compositions may alsocontain other active compounds providing supplemental, additional, orenhanced therapeutic functions. The pharmaceutical compositions may alsobe included in a container, pack, or dispenser together withinstructions for administration.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known to those of ordinary skill inthe art. The administration may, for example, be intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous ortransdermal. It may also be possible to obtain compositions which may betopically or orally administered, or which may be capable oftransmission across mucous membranes.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerin, propylene glycol, or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Suchpreparations may be enclosed in ampoules, disposable syringes ormultiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, and sodium chloride in the composition. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate, and gelatin.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For oral administration, the antibodies can be combined withexcipients and used in the form of tablets, troches, or capsules.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches, and the like can contain any of the followingingredients, or compounds of a similar nature; a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration may be accomplished, for example, throughthe use of lozenges, nasal sprays, inhalers or suppositories. Forexample, in case of antibodies that comprise the Fc portion,compositions may be capable of transmission across mucous membranes inintestine, mouth, or lungs (e.g., via the FcRn receptor-mediated pathwayas described in U.S. Pat. No. 6,030,613). For transdermaladministration, the active compounds may be formulated into ointments,salves, gels, or creams as generally known in the art. Foradministration by inhalation, the antibodies may be delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide or anebulizer.

In certain embodiments, the presently disclosed antibodies are preparedwith carriers that will protect the compound against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. Liposomal suspensions containingthe presently disclosed antibodies can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It may be advantageous to formulate oral or parenteral compositions in adosage unit form for ease of administration and uniformity of dosage.The term “dosage unit form” as used herein refers to physically discreteunits suited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of the composition of the inventioncan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. Compositions that exhibit large therapeutic indicesare preferred.

For any composition used in the present invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays.Examples of suitable bioassays include DNA replication assays, cytokinerelease assays, transcription-based assays, PD-1/PD-L1 binding assays,creatine kinase assays, assays based on the differentiation ofpre-adipocytes, assays based on glucose uptake in adipocytes,immunological assays other assays as, for example, described in theExamples. The data obtained from the cell culture assays and animalstudies can be used in formulating a range of dosage for use in humans.A dose may be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC₅₀ (i.e., theconcentration of the antibody which achieves a half-maximal inhibitionof symptoms). Circulating levels in plasma may be measured, for example,by high performance liquid chromatography. The effects of any particulardosage can be monitored by a suitable bioassay. The dosage liespreferably within a range of circulating concentrations with little orno toxicity. The dosage may vary depending upon the dosage form employedand the route of administration utilized.

The following Examples do not in any way limit the scope of theinvention. One of ordinary skill in the art will recognize the numerousmodifications and variations that may be performed without altering thespirit or scope of the present invention. Such modifications andvariations are encompassed within the scope of the invention. The entirecontents of all references, patents, and published patent applicationscited throughout this application are herein incorporated by reference.

EXAMPLES Example 1 Selection of PD-1 Binding ScFv's

An scFv phagemid library, which is an expanded version of the 1.38×10¹⁰library described by Vaughan et al. (Nature Biotech. (1996) 14: 309-314)was used to select antibodies specific for human PD-1. Soluble PD-1fusion protein (at 20 μg/ml in phosphate buffered saline (PBS)) orcontrol fusion protein (at 50 μg/ml in PBS) was coated onto wells of amicrotiter plate overnight at 4° C. Wells were washed in PBS and blockedfor 1 hour at 37° C. in MPBS (3% milk powder in PBS). Purified phage(1012 transducing units (tu)) was blocked for 1 hour in a final volumeof 100 μl of 3% MPBS. Blocked phage was added to blocked control fusionprotein wells and incubated for 1 hour. The blocked and deselected phagewere then transferred to the blocked wells coated with the PD-1 fusionprotein and were incubated for an additional hour. Wells were washed 5times with PBST (PBS containing 0.1% v/v Tween 20), then 5 times withPBS. Bound phage particles were eluted and used to infect 10 mlexponentially growing E. coli TG1. Infected cells were grown in 2TYbroth for 1 hour at 37° C., then spread onto 2TYAG plates and incubatedovernight at 30° C. Colonies were scraped off the plates into 10 ml 2TYbroth and 15% glycerol added for storage at −70° C.

Glycerol stock cultures from the first round of panning selection weresuperinfected with helper phage and rescued to give scFvantibody-expressing phage particles for the second round of panning. Atotal of two rounds of panning were carried out in this way forisolation of PD1-17, except in the second round of panning 20 μg/ml ofcontrol protein were used for deselection. Clones PD1-28, PD1-33, andPD1-35 were selected following three rounds of selection. Deselection inthe second and third rounds was carried out using 10 μg/ml controlfusion protein.

Antibodies to murine PD-1 were selected by soluble selection usingbiotinylated murine PD-1 fusion protein at a final concentration of 100nM. An scFv phagemid library, as described above, was used. PurifiedscFv phage (10¹² tu) in 1 ml 3% MPBS were blocked for 30 minutes, thenbiotinylated antigen was added and incubated at room temperature for 1hour. Phage/antigen was added to 250 μl of Dynal M280 Streptavidinmagnetic beads that had been blocked for 1 hour at 37° C. in 1 ml of 3%MPBS and incubated for a further 15 minutes at room temperature. Beadswere captured using a magnetic rack and washed 4 times in 1 ml of 3%MPBS/0.1% (v/v) Tween 20 followed by 3 washes in PBS. After the last PBSwash, beads were resuspended in 100 μl PBS and used to infect 5 mlexponentially growing E. coli TG-1 cells. Infected cells were incubatedfor 1 hour at 37° C. (30 minutes stationary, 30 minutes shaking at 250rpm), then spread on 2TYAG plates and incubated overnight at 30° C.Output colonies were scraped off the plates and phage rescued asdescribed above. A second round of soluble selection was carried out asdescribed above.

Example 2 Specificity of Antibodies for PD-1 by a Phage ELISA

To determine the specificity of antibodies for PD-1, a phage ELISA wasperformed against PD-1 fusion protein and control proteins. IndividualE. coli colonies from selection outputs were picked into 96 well platescontaining 100 μl of 2TYAG medium per well. M13K07 helper phage wasadded to a multiplicity of infection (moi) of 10 to the exponentiallygrowing culture and the plates incubated an additional 1 hour at 37° C.Plates were centrifuged in a benchtop centrifuge at 2000 rpm for 10minutes. The supernatant was removed and cell pellets were resuspendedin 100 μl 2TYAK and incubated at 30° C. overnight with shaking. The nextday, plates were centrifuged at 2000 rpm for 10 minutes andphage-containing supernatant from each well was transferred to a fresh96 well plate. Phage samples were blocked in a final concentration of 3%MPBS prior to ELISA.

Human or mouse PD-1 fusion protein and control fusion and non-fusionproteins were coated overnight at 4° C. onto 96-well microtiter platesat 0.5-2.5 μg/ml in PBS. After coating, the solutions were removed fromthe wells, and the plates blocked for 1 hour in 3% MPBS. Plates wererinsed with PBS and then 50 μl of pre-blocked phage were added to eachwell. The plates were incubated for 1 hour and then washed 3 times withPBST followed by 3 washes with PBS. To each well, 50 μl of a 1:5000dilution of anti-M13-HRP conjugate (Pharmacia, Peapack, N.J.) was added,and the plates incubated for 40-60 minutes. Each plate was washed threetimes with PBST then 3 times with PBS. Fifty μl of TMB substrate wasadded to each well, and the samples were incubated until colordevelopment. The reaction was stopped by the addition of 25 μl of 0.5 MH₂SO₄. The signal generated was measured by reading the absorbance at450 nm using a microtiter plate reader. Clones showing specific bindingto PD-1 fusion protein but not to control fusion proteins were thusidentified and confirmed.

Specificity data for the PD1-17 scFv is shown in FIG. 1A. Reactivity ofPD1-28, PD1-33, and PD1-35 scFv's with human PD-1 is shown in FIG. 1B(an IgG₁ control did not bind PD-1).

Example 3 Identification of Antibody Clones

PD-1-binding scFv E. coli clones were streaked out onto 2TYAG plates andincubated overnight at 30° C. Colonies from these plates were sequencedusing pCANTAB6 vector sequence oligos to amplify the V_(H) and V_(L)regions from the scFv clone. Unique PD-1 binding clones were assayed forneutralization of PD-L1 binding to PD-1 as described in Example 4.Sequence differences between scFv and IgG formats are due to changesintroduced by PCR primers during the conversion from scFv to IgG.

Example 4 Biochemical Binding Inhibition Assay and Screen

ScFv production was induced by addition of 1 mM IPTG to exponentiallygrowing cultures and incubation overnight at 30° C. CrudescFv-containing periplasmic extracts were obtained by subjecting thebacterial pellets from the overnight induction to osmotic shock. Pelletswere resuspended in 20% (w/v) sucrose, 50 mM Tris-HCl, pH 7.5, 1 mM EDTAand cooled on ice for 30 minutes. Cellular debris was removed bycentrifugation, and the scFv was purified by chromatography andbuffer-exchanged into PBS. Purified scFv's (PD1-17, PD1-28, PD1-33, andPD1-35) were tested for the ability to inhibit the binding ofbiotinylated human PD-L1 fusion protein to human PD-1 fusion proteinimmobilized on plastic in a 96 well microtiter plate assay. Binding ofbiotinylated PD-L1 fusion protein was detected with AMDEX-alkalinephosphatase, and the signal generated was measured by reading theabsorbance at 405 nm using a microtiter plate reader. Data was expressedas a percentage of the total binding and a titration of scFvconcentrations was tested to establish clone potency as calculated IC₅₀values. Clone potency data for the scFv and IgG antibodies is shown inTable 5.

PD1-F2 scFv was produced and purified as described above. Cellsexpressing murine PD-1 were added at 10⁵ cells/well in a final volume of100 μl to a poly-D-lysine-coated 96 well microtiter plate. Cells werecentrifuged and washed twice in PBS, then blocked with 300 μl 1% BSA inPBS for 1 hour at room temperature. Blocked cells were washed threetimes in PBST, prior to addition of 25, μl/well of assay buffer (0.05%BSA, 0.05% Tween 20 in Dulbecco's PBS) or sample, followed by 25 μl ofbiotinylated murine PD-L1 fusion protein at 300 ng/ml. Binding ofbiotinylated PD-L1 fusion protein was detected with Amdex alkalinephosphatase and signals read as described above. Potencies of PD1-F2scFv and IgG are shown in Table 6.

TABLE 6 Potency of Anti-PD-1 ScFv and IgG Antibodies Clone ScFv IC₅₀(nM) IgG IC₅₀ (nM) PD1-17 726 2.5 PD1-28 560 1.4 PD1-33 74 1.8 PD1-35 852.3 PD1-F2 28 1.0

Example 5 Conversion of ScFv to IgG

Heavy and light chain V regions from scFv clones were amplified by PCRusing clone-specific primers. PCR products were digested withappropriate restriction enzymes and subcloned into vectors containinghuman IgG₁ heavy chain constant domain (Takahashi et al. (1982) Cell 29,671) or vectors containing human lambda or kappa light chain constantdomains (Hieter et al. (1982) Nature 294, 536). Based on the germlinesof the V_(H) and V_(L) segments, it was determined whether kappa orlambda light chain constant domains were used for conversion (Table 7).

TABLE 7 Germlines of V_(H) and V_(L) Regions of PD-1 Antibody ClonesClone V_(H) germline V_(L) germline PD1-17 DP-70 DPL-8 PD1-28 DP-14DPL-23 PD1-33 DP-7 DPL-11 PD1-35 DP-65 DPL-2 PD1-F2 DP-47 L12 (κ)

The insertion of V region domains into plasmids was verified bysequencing of plasmid DNA from individual E. coli colonies. Plasmidswere prepared from E. coli cultures by standard techniques and heavy andlight chain constructs cotransfected into eukaryotic cells usingstandard techniques. Secreted IgG was purified using Protein A Sepharose(Pharmacia) and buffer-exchanged into PBS.

The binding affinity of the anti-mouse PD1 antibody PD1-F2 wasdetermined with a Surface Plasmon Resonance (SPR) system (BIAcore 3000)(Biacore, Piscataway, N.J.) using murine PD-1 fusion immobilized on aCM5 sensor chip. The concentration of PD1-F2 in the flow cell rangedfrom 7.81 to 125 nM, while the concentration of the anti-mouse PD1antibody J43 (eBioscience, San Diego, Calif.) ranged from 25 nM to 500nM. The equilibrium constant K_(D) for PD1-F2 is 6.7×10⁻⁹ M(K_(A)=1.5×10⁸ M⁻¹), whereas K_(D) for J43 is 3.8×10⁻⁷ M (K_(A)=2.6×10⁶M⁻¹).

The ability of anti-PD-1 IgG's to bind human or murine PD-1 wasdetermined as follows. ELISA plates were incubated with 2.5 μg/ml humanPD-1/IgG chimera overnight. Plates were washed with PBS/1% BSA andincubated with serial dilutions of a test antibody for 2 hours at roomtemperature (RT). After washing, saturating concentrations ofHRP-conjugated goat anti-human antibody or HRP-conjugated rabbitanti-murine antibody were added, and the samples were incubated for 1hour at RT. Unbound goat and rabbit antibodies were washed using PBS/1%BSA. The assay was developed using TBM. Results were expressed as OD 405absorbency values and are presented in FIGS. 2A-2C. Murine anti-humanPD-1 antibody J110 is commercially available (eBioscience, San Diego,Calif.) and was included for comparison.

Example 6 Selected PD-1 Antibodies Inhibit Binding of PD-L1 to PD-1

Inhibition assays were performed to assess the ability of the antibodiesto block binding of PD-L1 to PD-1. ELISA was performed as described inExample 2 with modifications. After incubation with a primary, anti-PD-1antibody for 2 hours at RT, a fixed concentration (1 μg/ml) ofbiotin-conjugated PD-L1-Ig was added, and the samples were furtherincubated for 1 hour at RT. After washing, saturating concentrations ofavidin-HRP were added, and incubated for 1 hour at RT. Unboundavidin-HRP was washed using PBS/1% BSA. The assay was developed usingTMB.

Results were compared to those obtained with J110 as shown in FIG. 3.Anti-human PD-1 antibodies J110 and PD1-30 did not inhibit the bindingof PD-L1 to PD-1. Anti-human antibodies PD1-17, PD1-28, PD1-33 andPD1-35 and anti-mouse antibody PD1-F2 block PD-1/PD-L1 interaction.

Example 7 PD-1 Antibodies Recognize Distinct Sites on PD-1

Inhibition assays were performed to map sites recognize by the varioushuman anti-human PD-1 antibodies. ELISA was performed as described inExample 6 with minor modifications. After incubation with primaryantibody for 2 hours at RT, a fixed concentration (0.25 μg/ml) ofbiotin-conjugated anti-PD-1 antibody J110 was added, and the sampleswere further incubated for 1 hour at RT. After washing, saturatingconcentrations of avidin-HRP were added, and incubated for 1 hour at RT.Unbound avidin-HRP was washed using PBS/1% BSA. The assay was developedusing TMB.

As shown in FIG. 4, binding of anti-human PD-1 antibodies (J110, J116,PD1-17, PD1-28, PD1-33, and PD1-35) defines at least two distinct siteson PD-1. Cross-blocking results show that J110 and J116, bind toidentical or overlapping sites while PD1-17, 28, 33, and 35 bind toanother distinct site. Binding of J116 or J110 to PD-1 blocks thebinding of J110. In contrast, binding of PD1-17, PD1-28, PD1-33, andPD1-35 do not block binding of J110. This suggests that the testedanti-PD-1 antibodies bind to at least two distinct epitopes: onerecognized by J110 and J116, and the other one recognized by PD1-17,PD1-28, PD1-33 and PD1-35.

Example 8 PD-1 Engagement Results in Decreased T Cell Responses

CD4+ T cells (5×10⁴ cells/well) were stimulated with tosyl-beads (Dynal,Great Neck, N.Y.) coated with anti-hCD3+/−PD-L1-Fc or anti-PD-1 (PD1-17or J110). Concentration of fusion protein or antibody titer was asindicated in the X-axis of FIG. 5. After 72 hours, proliferation wasdetermined by ³H-thymidine incorporation. Incorporated radioactivity wasdetermined using a LKB 1205 plate reader.

As shown in FIG. 5, PD-1 engagement by anti-PD-1 antibody PD1-17 orPD-L1.Fc caused a decrease in T cell proliferation. Thus, PD1-17 canmimic PD-1 ligands and delivered an inhibitory signal. As discussedbelow (Example 9), this inhibitory signal results in decreased T cellproliferation and IL-2 production. Antibodies PD1-28, PD1-33, and PD1-35have the same effect as PD1-17. The effect is dose-dependent, asactivation of cells in the presence of increasing concentrations ofPD1-17 or PD-L1.Fc results in decreased T cell proliferation. Thecontrol anti-PD-1 antibodies, J110 (FIG. 5) or J116 (data not shown), donot inhibit T cell responses and increasing the concentration of J110has minimal effect on T cell proliferation. For comparison, values arerepresented as percentage of the anti-CD3 response. “100%” representsCPMs obtained when cells were activated with anti-CD3/murine IgG-coatedmicrospheres. Altogether these results indicate that some but not allantibodies that recognize PD-1 can act as agonists of the PD-1 pathway.

Further experiments were performed to address whether PD-1downregulation of T cell responses required coordinate TcR/PD-1engagement on a single (CIS) or a separate (TRANS) cell surfaces. Twosets of microspheres were prepared: one set contained anti-CD3 andPD-L1.Fc (CIS), the other set contained anti-CD3 or PD-L1.Fc (TRANS).Inhibition through PD-1 was only observed under conditions where bothPD-1 and TcR were engaged by ligands on the same surface (CIS). At allbead:cell ratios tested, no inhibition was observed in conditions whereTCR and PD-1 signals were delivered on separate surfaces (TRANS).

To rule out steric hindrance in the TRANS experiments, similar assayswere set up using anti-CD3 antibody and B7.2.Fc. In these assays, B7costimulation of T cell responses was observed in both CIS and TRANSconditions. Altogether, these findings demonstrate that PD-1 proximityto TCR is required for the receptor modulator function on T cellactivation. Therefore, to modulate a T cell response, both activatingand inhibitory signals must emanate from the same surface whether thesurface is that of a cell or a bead.

Example 9 Blockage of PD-1 Engagement by Antibody Results in EnhancedProliferation

For assessing effect of soluble anti-PD-1 antibody on proliferation,CD4+ T cells were pre-activated for 48 hours withanti-CD3/anti-CD28-coated beads, harvested, and restimulated with theindicated concentration of PHA plus 10 ng/ml IL-2 in the presence ofPD1-17, J110, or control IgG. Each of the antibodies was added atvarious concentrations at initiation of the culture. Proliferation wasmeasured at 72 hr.

The results demonstrate that PD1-17 (FIG. 6) and PD1-35 (data not shown)enhanced proliferation of primary T cells. The control antibody J110 didnot enhance in vitro T cell responses. Selected anti-PD1 antibodies, asexemplified by PD1-17 and PD-35, inhibit the interaction of PD-1 withits natural ligands and thereby block delivery of a negative signal. Theblockade of the negative signal also results in enhanced proliferationand IL-2 production.

Example 10 Treatment of Disorders

Modulation of immune response regulated by PD-1 is useful in instanceswhere an immunosuppressive effect or augmentation of immune response isdesired. This example describes the use of PD-1 antibodies as PD-1agonists or antagonists to treat a subject at disease onset or having anestablished immune disorder or cancer, respectively.

Subjects at risk for or afflicted with cancer may be in need of immuneresponse augmentation would benefit from treatment with a PD-1antagonist, such as an anti-PD-1 antibody of the present invention in asoluble form. Most commonly, antibodies are administered in anoutpatient setting by weekly administration at about 0.1-10 mg/kg doseby slow intravenous (IV) infusion. The appropriate therapeuticallyeffective dose of an antagonist is selected by a treating clinician andwould range approximately from 1 μg/kg to 20 mg/kg, from 1 μg/kg to 10mg/kg, from 1 μg/kg to 1 mg/kg, from 10 μg/kg to 1 mg/kg, from 10 μg/kgto 100 μg/kg, from 100 μg to 1 mg/kg, and from 500 μg/kg to 5 mg/kg.

The antibodies are also used to prevent and/or to reduce severity and/orsymptoms of diseases or conditions that involve an aberrant orundesirable immune response, such as in autoimmune disorders exemplifiedbelow.

Multiple sclerosis (MS) is a central nervous system disease that ischaracterized by inflammation and loss of myelin sheaths. In theexperimental autoimmune encephalitis (EAE) mouse model for multiplesclerosis (Tuohy et al. (J. Immunol. (1988) 141: 1126-1130), Sobel etal. (J. Immunol. (1984) 132: 2393-2401), and Traugott (Cell Immunol.(1989) 119: 114-129), treatment of mice with a PD-1 agonist prior (andcontinuously) to EAE induction is expected to prevent or delay the onsetof MS.

Arthritis is a disease characterized by inflammation in the joints. Inthe collagen induced arthritis (CIA) mouse model for rheumatoidarthritis (Courtenay et al. (Nature (1980) 283: 666-628) and Williams etal. (Immunol. (1995) 84: 433-439)), treatment with a PD-1 agonist isexpected to prevent or treat rheumatoid arthritis (RA) or otherarthritic diseases.

Systemic Lupus Erythematosis (SLE) is an autoimmune diseasecharacterized by the presence of autoantibodies. The antibodies andcompositions of this invention can be used as PD-1 agonists to inhibitactivities of autoreactive T cells and B cells, and prevent or treat SLEor related diseases in NZB X NZW mice (a mouse model for SLE)(Immunologic Defects in Laboratory Animals, Gershwin et al. eds., PlenumPress, 1981) or in humans.

It is anticipated that PD-1 antibodies of the invention would beadministered as PD-1 agonists in ex vivo therapy with a frequency of oneper month or less. Treatment duration could range between one month andseveral years.

To test the clinical efficacy of antibodies in humans, individuals withmelanoma, prostate cancer, RA, SLE, MS, type I diabetes, are identifiedand randomized to a treatment group. Treatment groups include a placebogroup and one to three groups treated with a PD-1 agonist (differentdoses). Individuals are followed prospectively for one to three years.It is anticipated that individuals receiving treatment would exhibit animprovement.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification, all of whichare hereby incorporated by reference in their entirety. The embodimentswithin the specification provide an illustration of embodiments of theinvention and should not be construed to limit the scope of theinvention. The skilled artisan recognizes that many other embodimentsare encompassed by the claimed invention and that it is intended thatthe specification and examples be considered as exemplary only, with atrue scope and spirit of the invention being indicated by the followingclaims.

1. An antibody comprising the amino acid sequence as set out in SEQ IDNO:19, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, or SEQ ID NO: 52.2-14. (canceled)
 15. An antibody comprising human framework regions andmeans for specific binding to PD-1, wherein the antibody is capable ofblocking binding between PD-1 and PD-L1.
 16. The antibody of claim 15,wherein the means comprises a CDR derived from PD1-17, PD1-28, PD1-33,PD1-35, or PD1-F2.
 17. An isolated nucleic acid encoding the antibody ofclaim
 1. 18. An expression vector comprising the nucleic acid of claim17.
 19. A host cell comprising the vector of claim
 18. 20. (canceled)21. The nucleic acid of claim 17, wherein the nucleic acid encodes theamino acid sequence set out in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 47 or SEQ ID NO:
 49. 22. The nucleic acid of claim 21,wherein the nucleic acid comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 46 and SEQ ID NO:
 48. 23. A method of making an antibody thatspecifically binds with PD-1, the method comprises: (a) providing astarting repertoire of nucleic acids encoding a variable domain thateither includes a CDR3 to be replaced or lacks a CDR3 encoding region;(b) combining the repertoire with a donor nucleic acid encoding an aminoacid sequence substantially as set out in SEQ ID NO: 19, SEQ ID NO: 25,SEQ ID NO: 31, SEQ ID NO: 37, or SEQ ID NO: 52, such that the donornucleic acid is inserted into the CDR3 region in the repertoire, so asto provide a product repertoire of nucleic acids encoding a variabledomain; (c) expressing the nucleic acids of the product repertoire; (d)selecting an antigen-binding fragment specific for PD-1; and (e)recovering the specific antigen-binding fragment or nucleic acidencoding the binding fragment.
 24. An antibody produced by the method ofclaim
 23. 25. A method of modulating adaptive immune response comprisingcontacting a lymphocyte with an anti-PD-1 antibody.
 26. The method ofclaim 25, wherein the lymphocyte is a T cell, B cell, or monocyte. 27.The method of claim 25, wherein the antibody is as in claim
 1. 28. Themethod of claim 25, wherein the antibody is as in claim
 24. 29. Themethod of claim 25, wherein the antibody is immobilized on a supportmatrix or crosslinked.
 30. The method of claim 25, wherein the supportmatrix comprises one or more material chosen from agarose, dextran,cellulose, PVDF, silica, nylon, dacron, polystyrene, polyacrylates,polyvinyls, teflons, polyglycolic acid, polyhydroxyalkanoate, collagen,and gelatin.
 31. The method of claim 25, wherein the anti-PD-1 antibodymodulates immune cell response mediated by an antigen receptor.
 32. Themethod of claim 31, wherein the antigen receptor signal is co-presentedwith the anti-PD-1 antibody.
 33. The method of claim 31, wherein theantigen receptor signal and anti-PD-1 antibody are spaced by no morethan 100 μm.
 34. The method of claim 31, wherein the antigen receptorsignal is delivered by an anti-CD3 antibody.